Fluorescence Helps Measure Reactive Oxygen Species in Cigarette Smoke

The main hazard of smoking cigarettes is that it has been linked to major diseases: cancer, cardiovascular disease, emphysema and stroke among them. The connection between smoking and these diseases is oxidative stress, a condition in which lipids, proteins and DNA are damaged by the reactive oxygen species, such as nitric dioxide and peroxyl radicals, that are found in cigarette smoke.

To fully understand the effect that the reactive oxygen species have on smokers and to help find ways to reduce the harm caused by them, investigators must accurately measure their quantity within cigarette smoke. Typically, such measurements are performed using spin trapping combined with electron spin resonance or high-performance liquid chromatography. Unfortunately, this labor-intensive method is selectively reactive with some radicals and not sensitive at all to peroxyl radicals, which are the most prominent reactive species in cigarette smoke.

At Brunswick Laboratories LLC in Wareham, Mass., however, Dejian Huang and Boxin Ou developed a technique for quantifying reactive oxygen species in the smoke — and other flowing gases — using fluorescence.

The researchers tested several potential fluorescent probes for their reactivity to the smoke, including dihydrorhodamine 6G (DHR-6G), which frequently is used as a marker of oxidative stress in biological systems. When brought into contact with oxygen, DHR-6G loses two hydrogen atoms, becoming rhodamine 6G, a strongly fluorescent molecule.

They dissolved the DHR-6G and brought the solution into contact with cigarette smoke inside a standard laboratory bubbler. They used commonly available cigarettes obtained in the US and China as well as a reference cigarette produced by the University of Kentucky in Lexington, using an automatic smoking machine to produce the smoke.

By comparing the measured fluorescence intensity with that of off-the-shelf rhodamine 6G, the investigators calculated that the reference cigarettes each produced 384 nmol of reactive oxygen species. They also found that cigarettes from China produced consistently lower amounts (36 to 44 nmol per cigarette) than American brands (288 to 416 nmol each). A smokeless cigarette marketed in the US produced about 32 nmol. Chinese cigarettes typically are made of bright tobacco, whereas American ones are made of burley tobacco.

“Our results suggest that (the) tobacco leaves have an important effect on reactive oxygen species concentration,” Huang said. The composition of the leaves or the manner in which they are processed, he added, could feasibly be altered to lower the amount of reactive oxygen species in cigarette smoke.

The researchers note that DHR-6G does not directly react with either diluted hydrogen peroxide or lipid peroxides from the cigarette smoke, but that these substances are not as reactive as other radical species. They also acknowledge that DHR-6G is too expensive for large-scale measurements but say that a gas splitter could help reduce the amount of smoke that enters the measurement chamber.

They would like to miniaturize the whole system to lower the amount of the sample, DHR-6G and solvents that are required and, Huang said, “it would be great to have a multichannel system for parallel monitoring of several reactions at once.”

The group’s research is financed, in part, by Philip Morris USA in Richmond, Va.